Studies On The Reparation For NAFLD Model Of Polypeptides From Meretrix Meretrix

Non-alcoholic fatty liver disease,similarly with Alcoholic fatty liver disease,is a kind of disease caused for heredity,environment,and metabolism and has nothing to do with the alcohol.As the complex pathogenesis,Non-alcoholic fatty liver disease has no specific drug.Non-alcoholic fatty liver disease was mainly treated for Control of risk factors,exercise therapy,adjust the diet structure.In medication,the drugs usually used were metformin,pioglitazone,vitamin E,etc.Meretrix meretrix widely distributed in the coastal areas of our country,is an important economic mudflat shellfish resources.Meretrix meretrix tastes delicious and has rich nutrition including carbohydrate,protein and vitamin.Meretrix meretrix has much medicinal value,such as anticancer,antioxidant and anti-aging,is a kind of health food to be development.The effect of Meretrix meretrix to NAFLD cell models has not yet been reported.In this study,the Meretrix meretrix was regarded as the research object.Studied the repair effect on Non-alcoholic fatty liver disease for MMO and its mechanism of action.This study provide a new theoretical basis and practical basis for the research of liver protected medicine.Research contentsThe Chang liver cell NAFLD model and the mice NAFLD model were treated with MMO which was made the different concentrations of drugs.The following five experiments were conducted to explore the mechanisms for Chang liver cell NAFLD model and the mice NAFLD model.1.The Chang liver cell NAFLD model was made by Palmitic acid treated.The lipid droplet was observed with oil red staining and detected the Liver cell function indexes.2.The Meretrix meretrix was hydrolysis by pepsin,alkaline protease,Neutral,protease,papain and trypsin.The best hydrolysis was obtained by rthogonal test including temperature,time,doses,pH and feed solution proportion.The amino acid sequence wasdetected by ultra-filtration and HPLC and amino acid sequence detection.Detected the content of ALT,AST,?-GT,MDA and SOD after the MMO treated.3.oil red staining was used to observe the lipid droplet in Chang liver cell which was treated by MMO.The early apoptosis and the mitochondrial membrane potential was detected by flow cytometry.4.The expression of apoptosis-related proteins(Bax,Bcl-2,Caspase-9,Caspase-3,TNF-?,JNK)were detected by western blot analysis after the Chang liver cell was incubated with MMO.5.The mice NAFLD model was obtained and the morphology in mice liver was observed.Then detected the function indexes of mice and observe the morbidity of the mice liver for HE staining.Research results1.NAFLD cell model was established by treating with palmitic acid of 15 ?g/mL for 48 h.Liver function indexes detection results showed that ALT,AST,?-GT,MDA increased as the palmitic acid treated.Oil staining showed that the normal Chang liver cell has little lipid droplet and much lipid droplet in model group.2.The best hydrolysis condition on the basis of the index of TG content were: 40?,solid-liquid ration 1:2,pH 9.5,enzyme dosage 1000 U/g,time 8 h for Alcalase.The amino acid sequence was Gln-Leu-Asn-Trp-Asp.Liver function indexes detection results showed that ALT,AST,?-GT,MDA decreased with the MMO treated as SOD increased.3.The lipid droplet decreased with the medicine concentration increased.Annxin V-FITC/PI staining showed that the normal Chang liver cell have no much early apoptosis cell.The early apoptosis in model group increased and decreased after MMO treated.The early apoptosis decreased with the MMO concentration increased.After the Chang liver cell was incubated with different concentration of MMO,the result showed that the degree of decline in mitochondrial membrane potential decreased gradually with the drug concentration increasing using flow cytometry,which indicates that the apoptotic cells show the significant decrease trend.4.Western blot results demonstrated that each gene protein expression in Chang liver has a significant change after the MMO treated.With the concentration of MMO increased,the expression of Bax?Caspase-9?Caspase-3?JNK decreased,but the expression of Bcl-2 increased.The ratio of Bcl-2/Bax also increased significant,which induced the decline in mitochondrial polarity.5.Mice liver morphology can be observed that the model group mice liver were milky white,and damaged seriously.After the MMO treated,the mice liver got lighter and the damage got smaller.The results of morbidity of the mice liver for HE staining showed that the hepatic lobule of model group mice were disorder and the hepatic cord were broken.There have much cavitations in liver cells.The cavitations in the group which treated with MMO decreased and the hepatic cord got completed.Liver function indexes detection results showed that ALT,AST,?-GT,MDA decreased with the MMO treated as SOD increased.So,the MMO has a significant repair effect for the damaged liver of mouse.Conclusions1.The amino acids sequence of Meretrix meretrix oligopeptides was Gln-Leu-Asn-Trp-Asp.2.The MMO have a significantly repair effects on NAFLD model,and they show a significant dose relationship.3.The MMO have a significantly repair effects on NAFLD cell model.The mechanism was to up-regulating the protein expression of Bcl-2,down-regulating the protein expression of Bax,inhibition the protein expression of Caspase-3 and Caspase-9.MMO has a significant protection to NAFLD model.